The outer membrane (OM) of Gram-negative bacteria is an essential organelle and requires the proper assembly of its primary constituents, outer membrane proteins (OMPs), and lipids such as phospholipids and lipopolysaccharides, in order to prevent the entry of toxic small molecules into the cell. Although recent advances have identified some factors involved in targeting OMPs and lipids to the OM, the targeting pathway and mechanism(s) governing the coordination of OM assembly remain elusive. This application describes a powerful and innovative approach in which a combination of chemical genetics and conventional genetic and biochemical techniques allow for the identification of novel components of the OM assembly machinery(ies). Small molecules can be employed to probe for non-target suppressors of OM permeability defects and identify putative candidates of the assembly machinery for future characterization. Candidates found to be linked to both lipid and OMP assembly will be further investigated as potential coordinators of OM assembly. Crosslinking of tagged assembly candidates will determine localization of machinery components, and the extent of contact with unfolded OMPs. Furthermore, approaches outlined above promise to yield valuable insight into homologous mechanisms that drive mitochondrial membrane assembly in eukaryotic systems. [unreadable] [unreadable]